Delipidation of Membrane Fraction for HPLC Analysis

Hey All

I'm interested in analysing membrane proteins of various cell lines via
RP-HPLC. The plan was to mechanically lyse the cells, spin down the
membranes, solublise them and then do the analysis.

The problem is getting rid of all the lipid from the membranes. My guess
is if I inject all the lipid onto the column, all I'll succeed in doing
is binding a heap of muck to my column. I could do a TCA precipitation
of my proteins after the solubisation, problem being I'm trying to
preserve the oxidation/post translational modification state of my
proteins, and TCA precips seem cause any number of nasty reactions in my
proteins of interest.

Anyone got any bright ideas as to what I might try ??

Cheers
GregP

"Greg Pankhurst" <[Only registered users see links. ].unsw.edu.spamblock.au > wrote in
message
news:1fzj4ez.129n0zksj90qoN%g.pankhurst@spamblock. cfi.unsw.edu.spamblock.au...


I am sorry to be a bit sarcastic, but are there any papers on the subject
that would suggest the protocol? In addition, I believe many of membrane
proteins would be lipoproteins or the ones dependent on the membrane for
stability, thus you might be just better off trying to solubilize the
membrane fraction in organic solvents compatible with RP-HPLC, like
acetonitrile, acetone, etc. Also, you should always use a pre-column, so
that the expensive column will not get clogged by gunk. If you still prefer
water-based buffers, try digesting lipids with lipase. However, many
membrane proteins, again, might become unstable and would precipitate.
Anyway, I would go with organic solvents. I am sure you could find a lot on
the subject in Pubmed or in books. There is a number of methods books
specifically dealing with analysis of membrane proteins. And I must say,
this is a very broad and well studied area of biochemistry, so you might
need to look for information pertinent to your particular organism or type
of membranes.
-Emir

Greg Pankhurst wrote:


Removal of lipids from membrane proteins usually results in aggregation,
the resulting pellet is difficult if not impossible to redissolve.

The usual procedure is to solubilise the membranes in detergent (Triton,
C12E6, octyl-glucoside, dodecyl-maltoside or similar) and then use
normal chromatographic techniques to separate the
detergent/lipid/protein micelles. As long as you have enough detergent
present that each micell contains at most one protein molecule, this
works very well. Look for a couple of papers written by J.L. Rigaud for
technical details.

Note that RPC is not normally useful even for soluble proteins, as they
can not be eluted from the column again. Only peptides are amendable to
this technique. You may consider hydrophobic interaction chromatography
(HIC) instead, which uses phenyl- or butyl groups instead of octadecyl-
to limit the strength of interaction with the column, but for membrane
proteins even that may not suffice.